J. Mol. Biol. (1992) 223, 977-989

Bacteriophage Pl Genes Involved in the Recognition Cleavage of the Phage Packaging Site (pat)

and

Karen Skorupski, James C. Pierce, Brian Sauer and Nat Sternberg Du Pont Merck Pharmaceutical Co. Du Pont Experimental Station Wilmington DE 19880-0328, U.S.A. (Received 22 July

1991; accepted 7 October 1991)

The packaging of bacteriophage Pl DNA is initiated by cleavage of the viral Dh’A at a specific site, designated pat. The proteins necessary for that cleavage, and the genes that encode those proteins, are described in this report. By sequencing wild-type PI DNA and DNA derived from various Pl amber mutants that are deficient in pat cleavage, two distinct These genes appear to be co-ordinately genes, referred to as pacA and pacB, were identified. transcribed with an upstream Pl gene that encodes a regulator of late PI gene expression (gene IO). pacA is located upstream from pacB and contains the 161 base-pair pat cleavage site. The predicted sizes of the PacA and PacB proteins are 45 kDa and 56 kDa: respectively. These proteins have been identified on SDS-polyacrylamide gels using extracts derived from Escherichia coli cells that express these genes under the control of a bacteriophage T7 promoter. Extracts prepared from cells expressing both PacA and PacB are proficient for site-specific cleavage of the Pl packaging site, whereas those lacking either protein are not. However, the two defective extracts can complement each other to restore functional pat cleavage activity. Thus, PacA and PacB are two essential bacteriophage proteins required for recognition and cleavage of the Pl packaging site. PacB extracts also contain a second Pl protein that is encoded within the pacB gene. We have identified this protein on SDS-polyacrylamide gels and have shown that it is translated in the same reading frame as is PacB. Its role, if any, in pat cleavage is yet to be determined. Keywords:

bacteriophage

Pl;

DNA

packaging;

1. Introduction

t Abbreviations used: ds, double-stranded; bp, basepair(s); wt, wild-type; AMPR, resistance to ampicillin due to the bla gene in PAC plasmids; CMR, resistance to chloramphenicol due to the chloramphenicol transacetylase gene from transposon Tn9; IPTG, isopropyl -B-D-thiogalactopyranoside; kb, lo3 base-pairs. $03.00/O

site; pat cleavage;

terminase

ized from ds DNA phages typically exist in multimerit complexes composed of two subunits, one large and one small, encoded by distinct, usually adjacent, genes. The large and small terminase subunits (respectively) have been identified from phage lambda: gpA and gpNu1; phages T3 and T7: gp19 and gp18; phage T4: gp17 and gpl6; and phage P22: gp2 and gp3 (see Black, 1989). Following an initial cut in the DNA concatemers by terminase, DNA packaging in ds DNA phages proceeds unidirectionally from the cut end of the molecule into the prohead. A second cleavage then separates the packaged DNA from the rest of the concatemer and generates a new end that serves as the starting point for a second round of DNA packaging. Since several rounds of DNA packaging occur before the whole process reinitiates, this mode of packaging of DNA is described as processive. The strategies used by the terminase enzymes for cleavage of DNA during packaging varies somewhat among the different phages. For lambda, T3 and T7,

An important step in the maturation of many tailed, double-stranded (dst) DNA bacteriophages is the processing of concatemerized phage DNA into lengths of genome size or slightly larger and the packaging of that DNA into phage particles. Specific DNA dependent ATPases, referred to as terminases or pacases, generate the ends of mature phage DNA and are involved in the translocation of DNA into empty capsids during packaging (for recent reviews, see Casjens, 1985; Black, 1989). Generally, these enzymes are not physically associated with either the prohead or the mature phage particle. The terminases that have been character-

0022-2836/92/040977-13

packaging

977 0 1992 Academic E’ress Limited

978

K. Skorupski et al.

each cleavage made by the terminase in the DNA concatemer occurs at a specific DNA sequence (Hohn, 1983; Hashimoto & Fujisawa, 1988). Thus, the mature DNA in each virion contains identical ends. For T4, cleavage of DNA concatemers appears to be completely non-sequence-dependent (Streisinger et al., 1967); the initial cleavage is apparently random and the second cleavage occurs when the prohead has been completely filled with DNA (the headful cut). This method of packaging generates virions containing terminally redundant, circularly permuted DNA molecules. Evidence indicates that an endonuclease distinct from the terminase enzyme may be responsible for carrying out the headful cleavage reaction. An unusual feature of this endonuclease is its location within the phage prohead (Rao & Black, 1985). For P22 and PI, the initial cut in the DNA concatemer occurs at a specific sequence, designated pat, and the headful cleavages are generated non-specifically when the prohead is completely filled (Tye et al., 1974; Bachi & Arber, 1977). It is not clear if the same enzyme performs both of these cleavages. Several sequence-dependent terminase cleavage sites have been characterized in detail. These sites lie either within or close to the genes encoding the terminase proteins and have a bipartite structure composed of separate terminase binding and cutting sites. The lambda cos site is about 200 base-pairs (bp) in size and consists of specific terminase nicking (cosN) sites flanked asymmetrically by binding (cosB) sites (for a review, see Becker & Murialdo, 1990). Nicks introduced at cosN by the terminase produce 12-base single-stranded 5’-protruding ends. The P22 packaging site is approximately 120 bp in size. Cleavage within this region is imprecise and generates blunt ends that are separated by about 20 bp (Casjens & Huang, 1982; Backhaus, 1985). A ten-bp pat recognition sequence has been identified (Casjens et al., 1987) and it has a central location within the cleavage sites. The Pl pat site is 161 bp in size, and it is cleaved within a centrally located 13 bp region to generate protruding two-bp 3’ termini (Sternberg & Coulby, 1987b). The flanking regions of the site contain four hexanucleotide elements (5’-TGATCA/G) on one side and three on the other. These elements are necessary for efficient cleavage and represent recognition and binding domains for the Pl pacase enzyme (Sternberg & Coulby, 19876; N. Sternberg, unpublished results). Each hexanucleotide element is also a Dam methylation site (5’-GATC) and methylation of at least some of these elements is necessary for pat cleavage (Sternberg & Coulby, 1990). Little is known about the Pl pacase enzyme. Analysis of five Pl amber mutants that are unable to cleave the pat site has defined a gene (gene 9) essential for pat cleavage (Sternberg & Coulby, 1987a). In order to characterize this gene in greater detail and to determine precisely the locations of the amber mutations, DNA from the pat region of both wild-type (wt) Pl and three of the five amber mutants was sequenced. We present here the

nucleotide sequence of this region and show that the amber mutations define two genes essential for pat cleavage: gene 9 and a previously unidentified gene immediately downstream from gene 9. The 161-bp packaging site lies completely within the coding sequence of gene 9 (now designated pacA). Both pacA and the downstream gene, pacB, appear to be co-ordinately expressed from an early Pl promoter that is located immediately upstream of Pl gene 10. The pacA and pacB genes have been cloned into expression vectors and their gene products have been identified. Both PacA and PacB proteins are necessary for cleavage of the Pl pat site.

2. Materials and Methods (a) Bacteria,

phuge and plasmids

The hosts for expression of the pPAC plasmids were coli BL21(DE3) (F- hsdS gal (iclts857 indl Sam7 nin5 lacUV5T7 genel); Studier & Moffatt, 1986) trpC22 : : TnlO and its derivative KSKll (BL21(DE3) supF), which was produced by Pl transduction from BS503 (Y1090 r-m-; Huynh et al., 1985). The E. coEi strain used for producing extracts containing pacase activity was NS3208 (sup” recD hsdR mcrA mcrB (Pl Cm-2 cl.100 amlO. r-m-); Sternberg et aE., 1990). Other E. coli strains used were W3350 (sup”) (Campbell & Balbinder, 1958), K175 (supD) (Scott, 1968), YMC (supF) (Dennert & Henning, 1968) and CA273 (supG) (Stretton et al., 1966). Pl phage defective for pat cleavage contained either the amber 9.16, amber 43 or amber 131 mutations (Sternberg & Coulby, 1987a; Walker & Walker, 1983) in super-virulent (vi?) backgrounds (Scott, 1968) or in backgrounds containing the temperature-sensitive repressor mutation cl.100 (Rosner, 1972). Pl DNA from the pat region (map units 950 to 990) was cloned into either Ml3 mp18 and mp19 or pUC18 and 19 (Messing, 1983) for sequencing and for oligonucleotide-directed mutagenesis. The pPAC plasmids constructed in this work (see Fig. 2) contain DNA from the pat region of Pl cloned into the StuI and/or BamHI sites of the transcription vector pET7 (Rosenberg et al., 1987). Pl EcoRI fragment 20, which contains the phage pax site and was used as a substrate in the in vitro pacase assay, was generated by EcoRI digestion of pIJC18: EcoRI-20 (Sternberg & Coulby, 1990). The E. coli PI lysogens constructed in this work are: KSKOB (K175 pPAC1 (Pl Cm-2 cl.100 am9.16 r-m-)); and KSK07 (K175 pPAC4 (Pl Cm cl.100 am43 r-m-)). Escherichia

(b) Media L broth, L agar plates and top agar, LCA plates (L agar plus 2 mM-CaCl,) and M63 minimal broth were prepared as described (Silhavy et al., 1984). When necessary, ampicillin was used at a concentration of 25 pg/ml in liquid cultures and 150 pg/ml in plates and chloramphenicol was used at a concentration of 25 pg/ml in liquid cultures and in plates. (c) DNA

methods

Plasmid DNA was isolated either by lysis with alkali (Sambrook et al., 1989) or by cesium chloride densitygradient centrifugation (Davis et al., 1980). Pl DNA was prepared as described by Sternberg et al. (1977) and single-stranded Ml3 DNA as described by Messing (1983). DNA fragments were purified from agarose gels by

PI Genes Involved in Pl pat Cleavage electroelution (IBI apparatus). DNA transformation, ligation, polynucleotide kinase and single-stranded fill-in reactions were all carried out as described by Sambrook et al. (1989). Oligonucleotides were synthesized on a Pharmacia-LKB Gene Assembler Plus using phosphoramidite chemistry and were used without further purification. Single and double-stranded DNA was sequenced using the dideoxy chain termination method (Sanger et al., 1977) using primer extension reactions (see Fig. 1) with [35S]dATP (NEN; 1300 Ci/mmol). The reactions were fractionated on 8% polyacrylamide gels and visualized by autoradiography. All DNA sequence information was obtained by sequencing both complementary strands and regions of sequencing gels difficult to interpret (usually G + C-rich) were re-examined using dITP as a replacement for dGTP in the sequencing reaction (Barnes et al., 1983; Gough & Murray, 1983). Phosphorothioatebased oligonucleotide-directed mutagenesis (Sayers et al., 1988). was performed according to the protocol supplied by Amersham and the presence of the appropriate mutation was verified by DNA sequencing. (d) Transfer

of amber mutations

from

PI to plasmids

E. coli strains KSK02 and KSK07 were grown in L broth to A,,,,, = 0.6 at 3O”C, induced at 42°C for 15 min, and then incubated at 38°C for 90 to 180 min. The resulting lysates were used to infect strain W3350 and CMR) ampicillin-chloramphenicol-resistant (AMPR colonies (which presumably contain Pl : : pPAC cointegrate plasmids) were selected at 30°C on L plates. Several isolates were grown at 30°C to mid-log phase in L broth with ampicillin, plated at 42 “C on L AMP agar plates, and subsequently tested for CM sensitivity. AMPR, CMS colonies (which presumably have lost the Pl portion of the cointegrate by homologous recombination) were then tested by marker rescue with Pl wir’ am9.16 and Pl vi? am43 phages to verify the presence of the appropriate amber mutations on the pPAC plasmids. The am131 mutation in pPAC9 was introduced into that plasmid on a BglII fragment that was derived from Pl am131 DNA. The fragment extends from nucleotide 1234 (Fig. 1) to a BglII site in the Pl cl gene.

(e) Transfer of the Nhe amber mutation PI Cm cl.100 r-m-

from

pPAC16

to

Plate lysates of Pl Cm cl.100 r-m- were prepared on strain BL21(DE3) containing plasmid pPAC16. which carries the Nhe amber mutation, essentially as described by Walker & Walker (1975). The resulting lysates were used to infect strain CA273 and AMPR CMR colonies containing Pl : : pPAC cointegrate plasmids were selected at 30°C. Isolates were then grown in L broth at 30°C and induced at 42°C as described above. The Pl phage in these lysates carrying the Nhe amber mutation were identified by isolating CMR AMPs lysogens of strain CA273 unable to produce a zone of clearing when toothpicked onto a sup” lawn at 42°C. The presence of the mutation was confirmed by digesting Pl DNA with NheI. (Q Labeling

proteins

expressed from plasmid

constructs

The labeling procedure is a variation of that used by Studier & Moffatt (1986). Briefly, BL21(DE3) or KSKll containing the various plasmid constructs were grown in M63 media supplemented with 64% glucose and 61 y0 yeast extract to an A,,, of approximately 1.0. The cultures were then induced with 0.4 mM-isopropyl-/?-n-

979

thiogalactopyranoside (IPTG) and incubated at 37°C for 30 min. Rifampicin then was added to a concentration of 200 pg/ml. Incubation was continued an additional 60 min (for the 1.5 h sample) or for an additional 120 min (for the 25 h sample). At the time of sampling, 50 ~1 portions of the cultures were removed, labeled for 5 min with 10 pCi of [35S]methionine (NEN; 1000 Ci/mmol) and then pelleted for 5 min at 16,000 g. The cells were washed with 10 mm-Tris . HCl (pH 68), repelleted, and resuspended in 15 ~1 of cracking buffer. Samples were fractionated on 10% polyacrylamide gels (Laemmli, 1970) and visualed by autoradiography. (g) Preparation

of cell extracts for in vitro pat assay

Cell extracts from strain NS3208 were prepared as described in Sternberg (1990) for strain NS2962 except that a Kontes micro-ultrasonic cell disrupter was used (setting 4, 4 intervals of 30 s) to break t,he cells. Cell extracts from BL21(DE3) containing the various plasmid constructs were prepared in the same way except that the cells were grown to an A,,, of 1.0 at 37°C and induced with 0.8 mM-IPTG for 1 to 3 h prior to sonication. The extracts resulting contained approximately 10 mg protein/ml. (h) in vitro pat assay The substrate for this assay was prepared by first digesting pUC18 : EcoRI-20 DNA with EcoRI. The DNA was treated with calf intestinal phosphatase to remove the terminal phosphate groups and kinased with T4 polynucleotide kinase and [Y-~‘]ATP (NEN; 6000 Ci/mmol). The resulting concentration of substrate DNA was approximately 10 pg/ml and contained approximately 5.0 x lo6 cts/min/pg. This substrate (1 ~1) was added to each reaction containing 25 miw-Tris . HCI (pH 8-O), 25 mM-NaCl, 10 m&r-MgCl,, 62 mM each dTTP, dCTP, dATP, dGTP, 1 mM-ATP, 2 mM-dithiothreitol and 1 ~1 of each cell extract prepared as described above. The reactions were incubated at 30°C for 5 min and then were treated with 0.2% SDS and 100 fig proteinase K/ml for 30 min at 37°C. Samples were heated at 70°C for 10 min, fractionated on a 5% polyacrylamide gel, and visualized by autoradiography. (i) Sequence analyses DNA sequence information was analyzed with the aid of the Wisconsin GCG programs (Devereux et al., 1984). Consensus sequence searches of translated sequences were performed with the MacPattern program (EMBL Data Library, Heidelberg, Germany).

3. Results (a) Nucleotide sequence of the PI pat region: identi$cation of open reading frames Figure 1 shows the sequence of 2981 bp of Pl DNA starting at the EcoRI site within gene 10 (the junction of the Pl EcoRI-6 and EcoRI-20 fragments) and extending into the coding region of the cl gene?. Two open reading frames were identified t The EMBL/Genbank pat region is M74046.

accession number for the Pl

980

K. Skorupski

et al.

Figure 1. The nucleotide sequence of the Pl pat region. The upper portion shows a map of approximately 4 kb (1 kb is IO3 base-pairs) from the pat region of Pl DNA (not to scale) that contains genes 10, pacA. pacB and cl. The lower portion shows the nucleotide sequence of 2981 bp of this DNA that was generated using the strategy shown above the map. The rightward-pointing arrows beneath the map and within the DNA sequence indicate the direction of transcription through genes 10, pacA and pacB, and the leftward-pointing arrows show the direction of transcription through the cl gene. Within the sequence, the positions of the stop codons at the ends of genes 10 and cl are indicated. Putative ribosome-binding sites of the pacA and pacB genes are designated by a. double underline. Nucleotide changes within pacA and pacB produced by the various amber mutations are shown above the wt sequences and the mutation designations are indicated by small boxes beside the changes. The pat site is shown by a line above the nucleotide sequence (nucleotides 456 to 618) and the positions of the methyl&ion domains within this site are underlined. The pat cleavage site is contained within the box in the center of pat. The positions of relevant restriction sites are shown both in the map and in the sequence: E, EcoRI; H2, HincII, R, BgZII; N, NotI; H3, HaeTII; S, SmaI.

981

Pl Genes Involved in PI pat Cleavage Table 1 Molecular weights of PI proteins produced from PAC plasmids? Protein --____ PacA

.~

PXH

Pac(’ PacA-truncated PacA amber 9.16 PacA amber She PacB amber 131 Pa& amber 43 Pac(! amber 43

Predicted molecular mass (kDa)

Relative molecular mass

45.2

52

,556

54

38.4: 258 82 54 4.55

20 38 f 9 41 0

Plasmids pPAC pPAC pPAC pPAC pPAC pPAC pPAC pPAC pPAC

t Shown in Fig. 2. 2 The molecular weight of this protein was determined by sequencing the junction the pack coding sequence and the pSR322 nucleotide sequence. 5 Not detected on SDS-polyacrylamide gel in Fig. 3.

within this region. The upstream open reading frame, designated pacA, is 1191 bp in length and the downstream reading frame, designated pacB, is 1482 bp in length. The initiation codon for the paeA gene is a GTG codon 85 bp downstream from gene 10 (nucleotide 266 in Fig. 1). A near-consensus ribosome binding site is located six bp upstream from this GTG start site. Although an in-frame ATG codon lies 192 bp downstream from the GTG codon, this ATG codon is not the start of the PacA protein since an amber mutation positioned between the GTG and ATG codons destroys the production of the PacA protein (see below). The pacA termination codon (TAA) overlaps the ATG initiation codon for pacB. A poor ribosome-binding site is located five bp upstream from the pacB start codon. The cl gene, which is transcribed in a direction opposite to that of the pacA and pacB genes (Osborne et al., 1989), lies 24 bp downstream from pacB. The Pl pat site, which has been described previously (Sternberg & Coulby, 39873), is located entirely within the 5’-terminal portion of the pacA gene. We sequenced the pat region from three different Pl amber mutants defective in pat cleavage (Sternberg & Coulby, 1987a). These amber mutants, 9.16, 131 and 43, were previously assigned to a single cistron, gene 9, based on their inability to complement each other. Our results show that these mutations define two distinct genes. All three mutations are C to T transitions that convert glutamine codons to amber (TAG) stop codons. The amber 9.16 mutation lies within pacA at nucleotide 941 (amino acid residue 226) and the amber 131 and 43 mutations lie within pacB at nucleotides 1597 and 2668 (amino acid residues 47 and 404), respectively (Fig. I).

(b) Identification

of the proteins encoded by the pacA and pacB genes

We have used the T7 transcription vector pET7 (Rosenberg et al., 1987) to facilitate the identification of the proteins encoded by the pacA and pa&B

-~.-

2. 3. 10 3.4 3. 4, 9. 13 1 5 16 9 6 6

created between

genes. Pl fragments of DNA were cloned into pET7 immediately downstream from the bacteriophage T7 gene 10 promoter and transcription of the DNA was initiated by derepression of the T7 RNA poiymerase gene (controlled by the ZacUV5 promoter) on a resident lambda prophage (DE3; Studier & Moffatt, 1986). After induction of the T7 RNA polymerase gene with IPTG, it is usually sufficient to visualize gene products produced from the PETS vector by Coomassie blue staining. In the case of PacA and PacB, however, the visualization of required pulse-labeling with protein either [35S]methionine in the presence of rifampicin in order to reduce the expression of host genes. The pET7 vector does not contain a transcription terminator downstream from the cloned genes to prevent transcription from the T7 promoter into the bla gene (Rosenberg et al., 1987). Thus, /?-lactamase is expressed along with the cloned gene products in this system and serves as a useful control for the effectiveness of induction. Mature b-lactamase has a M, value of 28,800 and migrates slightly faster than its unprocessed 3 1,300-M, precursor. Several plasmids were constructed to visualize the proteins encoded by the pacA and pacB genes (Fig. 2, Table 1). The construct pPAC3 produces both the PacA and PacB proteins with apparent M, values of 52,000 and 54,000, respectively (Figs 3(a) and (b), lanes 3 and 4). The predicted sizes of these proteins, determined from their nucleotide sequences, are 452 kDa for PacA and 55.6 kDa for PacB. The construct pPACl0 produces only the 52,000-n/r, PacA protein (Fig. 3(a), lanes 5 and 6) and the construct pPAC4 produces only the 54,000-M, PacB protein (Fig. 3(b), lanes 5 and 6). To verify that the 52,000 and 54,000-M, proteins produced with the above plasmids were indeed PacA and PacB, an additional series of constructs was generated that should produce truncated proteins (Fig. 2, Table 1). Truncated PacA proteins were produced either by the removal of the 3’ end of the gene (plasmid pPAC1) or by the introduction of the 9.16 amber mutation into the gene (plasmid pPAC5). In both of these cases, the wt PacA protein

982

et al.

K. Skorupski

pm- cleavage site

c 1 repressor binding site pPAC3

pPAC2 amber

pPAClS

Nhe

w

pPACl0

pPAC1

-

pPACS

-

amber 9.16

pPAC4 *amber 43 pPAC6 *amber

131

pPAC9 pPAC13

-

Figure 2. A map of approximately 4 kb from the pat region of Pl DNA showing the extent of DNA contained within each of the PAC plasmids used in this work (not to scale). Open boxes show the positions of the genes IO, pa&, pa&?, cl (3’ end) and the putative pa& gene. The large rightward-pointing arrow shows the position of the early promoter upstream from gene 10 and its direction of transcription. The small leftward-pointing arrow shows the direction of transcription through the cl gene. The shaded box shows the position of the cl repressor-binding site which overlaps the gene 10 promoter and the filled box shows the position of the pat site within the pad gene. The positions of the amber mutations in the constructs pPAC5, pPAC6, pPAC9 and pPAC16 are indicated by asterisks. The positions of relevant restriction sites are shown: E, EcoRI; H2, Hⅈ B, BgZII; N, NotI; H3, HaeIII; S, SmaI. The Pl proteins produced by the PAC plasmids and their molecular weights are shown in Table 1.

was not produced

from

the constructs.

The protein

produced from pPAC1 is a 38,000-M, product (Fig. 3(a), lanes 7 and 8) and is the size expected from the DNA sequence (Table 1). The 9.16 amber fragment produced from pPAC5 cannot be visualized in Figure 3(a), lanes 9 and 10, presumably because it migrates to a position in the gel containing other proteins. Truncated PacB proteins were produced by introducing either the 43 (plasmid pPAC6) or 131 (plasmid pPAC9) amber mutations into the gene. As expected,

neither

of these plasmids

produced the wt PacB protein. The protein produced by pPAC6 has a iM, of 41,000 (Fig. 3(b), lanes 7 and 8) and is similar to that expected from the DNA sequence (Table 1). The protein produced by pPAC9 should be 54 kDa in size and is too small to be seen in the gel in Figure 3(b), lanes 9 and 10).

There amounts

is a rather of the PacA

significant and PacB

reduction in the proteins produced from the construct that expresses both pacA and pacB (pPAC3, Fig. 3(a) and (b), lanes 3 and 4)

compared to the plasmids that express either of these genes alone (pPAC10, Fig. 3(a), lanes 5 and 6 and pPAC4, Fig. 3(b), lanes 5 and 6). Moreover, when both proteins are made together, much less appears to be detectable at 25 hours than at 1.5 hours (compare lane 3 to lane 4 in both Fig. 3(a) and (b)). We have also observed a reduction in the amount of pPAC3 plasmid DNA after induction with IPTG (data not shown). These results suggest that simultaneous expression of the PacA and PacB proteins causes the plasmid from which they are being expressed to be cleaved, thereby preventing further expression of the pat genes. This interpreta-

PI Genes Involved in Pl pat Cleavage

Pace PacA

983

3 f

42 ,OOO- M, protein

tPacA

truncated

-+P-lactamase *Bloctamase

(precur (mature

-3or) !)

PacC

Pace ic PocA + c

Poc6 43 amber

+P-Ioctomase

(precursor)

+‘p-

(mature)

loctamase

+PacC

(b) Figure 3. Identification of the proteins encoded by the pacA and pa& genes. Cultures of BL21(DE3) (sup”), or KSKll (supF) where indicated, earryin different plasmids (see Fig. 2 and Table 1) were grown in MB3 medium, induced with IPTG, and then labeled with [’ BSlmethionine 1.5 and 2.5 h after induction as described in Materials and Methods. The proteins were fractionated by electrophoresis in the presence of SDS on a 10% polyacrylamide gel with a 5% stacking gel. and detected by autoradiography. For each plasmid set, the 1st lane is the 1.5 h sample and the 2nd lane is the 2.5 h sample. (a) lanes 1 to 12, strain BL21(DE3); lanes 13 and 14, strain KSKll; 1 and 2, pET7; 3 and 4, pPAC3; 5 and 6, pPACl0; 7 and 8, pPAC1; 9 and 10, pPAC5; 11 to 14 pPAC16. (b) Lanes 1 to 12, strain BL21(DE3); 1 and 2. pET7; 3 and 4, pPAC3; 5 and 6, pPAC4; 7 and 8, pPAC6, 9 and lo, pPAC9; 11 and 12, pPAC13. tion is supported by the observation that both PacA and PacB are necessary for cleavage of the pat site (see below). Since the bla gene is located on these plasmids, this model also explains the reduced amounts of fi-lactamase detected in these constructs, especially in the 2.5 hour sample.

(d) Determination

of the pacA start site

The start of the pacA open reading frame appears to be a GTG codon 85 bp downstream from the termination codon of gene 10 (Fig. 1). This GTG codon is preceded by a near-consensus ribosome

984

K. Skorupski

binding site six bp upstream. The nucleotide sequence of the pacA open reading frame shows that an in-frame ATG codon lies 192 bp downstream from this GTG codon. Although no obvious ribosome-binding site precedes the ATG codon, the possibility that this codon serves as the start of the PacA protein cannot be ruled out a priori. To determine which of these two codons is the actual pacA start, an amber mutation was introduced into the pacA coding sequence 36 bp upstream from the ATG codon by oligonucleotide-directed mutagenesis. This mutation, an A to T transversion, changes a lysine residue to a termination codon and simultaneously creates a unique NheI site in the pacA gene, hence the designation Nhe amber. If the ATG codon is the actual start of the pacA gene, a plasmid carrying the Nhe amber mutation should produce the 52,000-M, PacA protein as seen with constructs pPAC3 and pPACl0 (Fig. 3(a), lanes 3 to 6). Alternatively, if the GTG codon is the pacA start site, the Nhe amber-containing plasmid should not produce the 52,000-M, protein. The size of the amber fragment produced from this construct is predicted to be 6.2 kDa in size (Table 1) and should not be detectable on a 10% polyacrylamide gel. When BL2l(DE3) carrying the plasmid with the LVhe amber mutation (designated pPACl6) is induced in a manner identical with that described above for the other pET7 plasmid constructs, the 52,000-M, PacA protein is not produced (Fig. 3(a), lanes 11 and 12). The inability of pPACl6 to produce the 52,000-M, PacA protein is due to the presence of the amber mutation, since the production of the protein is restored when the mutation is suppressed in strain KSK 11 (supF) (Fig. 3(a), lanes 13 and 14). Furthermore, plasmid pPACl6 does not produce functional PacA protein in a nonsuppressing strain (see below) and when the Nhe amber mutation is transferred to Pl, the mutant phage cannot form plaques on a strain lacking a suppressor mutation. These results indicate that the GTG codon is the actual start of the pacA gene. A protein with an apparent molecular mass of 42,000 Da is produced with construct pPACl6 (Fig. 3(a), lanes 11 to 14); this protein is also produced with constructs pPAC3 and pPACl0 (Fig. 3(a), lanes 3 to 6), but not with any of the other constructs in Figure 3(a). Since all of the constructs producing this protein contain the complete wt pacA gene, it is tempting to speculate that this 42,000-M, protein is the result of an internal start at the ATG codon that lies within pacA. The predicted size of a protein initiating at this ATG codon would be 37.7 kDa. (e) Identi$cation of a second Pl protein that is encoded within the pacB gene Close examination of the proteins from the plasmids pPAC3 (Fig. 3(a) and (b), lanes 3 and 4), pPAC4 (Fig. 3(b), lanes 5 and 6) and pPAC9 (Fig. 3(b), lanes 9 and lo), shows that a protein with a size of about 20,000-n/r, is produced by these from cells this protein is absent constructs;

et al containing either pET7 alone (Fig. 3(a) and (b), lanes 1 and 2) or from the pPACl0, pPAC1, or pPAC5containing cells (Fig. 3(a). lanes 5 to 10). The presence of this protein in extracts from pPAC4 (containing the complete wt pacB gene; Fig. 3(b). lanes 5 and 6) and extracts from pPAC9 (containing the 5’ pacB amber 131 mutation; Fig. 3(b), lanes 9 and lo), but not in extracts from construct pPAC6 (containing the 3’ pacB amber 43 mutation; Fig. 3(b), lanes 7 and 8) suggests both that this 20,000-M, protein is encoded by the C-terminal end of the pacB gene and that it is translated from the same reading frame as is the PacB prot’ein. Inspection of the DNA sequence supports this interpretation: no other open reading frame in the C-terminal portion of PacB is sufficiently large t,o encode a 20 kDa protein. Plasmid pPACl3 (Fig. 2) was constructed to define better the limits of the 20 kDa coding region within pacB. pPACl3 contains a DNA fragment that extends from a HaeIII site within pacB at nucleotide 2390 (Fig. 1) to a HaeIII site within cl. This plasmid also produces the 20,000-M, protein, henceforth called PacC (Fig. 3(b), lanes 11 and 12). Since the 43 amber mutation in pPAC6, which destroys the production of the 20,000-M, PacC protein, is only 278 bp downstream from the pacH HaeIII site, translation of this protein must initiate within this 278 bp region. Four ATG codons within this region could serve as potential start sites for the PacC protein. The first of these is at’ nucleotide 2428 and is the most likely start site, since it would encode a protein of 19.3 kDa. The other ATG codons are at nucleotides 2560, 2611 and 2647 and would encode significantly smaller proteins of 14.6, 12.4 and 10.9 kDa respectively. (f) The PacA

and PacB proteins function cleave the pat site

in vitro

to

Since the genetic evidence indicates that pacA and pacB are two essential genes involved in pat cleavage in vivo, it was of interest to determine whether or not the proteins encoded by these genes function in vitro to cleave the Pl pat site. The in vitro cleavage assay used is a variation of the stage 1 in vitro packaging reaction described by Sternberg (1990). The substrate for this assay is a 687 bp [32P] end-labeled fragment containing the PI pat site (Fig. 4(a)). Cleavage of this fragment at pat produces two smaller fragments of 545 bp and 142 bp (Fig. 4(b)). Cell extracts prepared by the induction of BL21(DE3) strains containing plasmids that express pacA and pacB either alone or together were tested for their ability to cleave the 687 bp fragment’. Extracts prepared from cells expressing both pacA and pacB together (pPAC3; Fig. 4(b), lane 3) cleave the substrate at an efficiency comparable to that of an extract prepared from an induced Pl lysogen (Fig. 4(b), lane 2). In contrast, extracts prepared from cells expressing either pacA or pacB alone do not cleave the substrate (pPAC2; Fig. 4(b), lane 4 and pPAC4; Fig. 4(b), lane 5). However,

985

Pl Genes Involved in PI pat Cleavage pat-cleavage

site

; 545 bp

PSZ 142 bp

(a 1

687 bp uncut fragment -* 545 bp cleavage

fragment

+

142 bp cleavage fragment

+

lb)

Figure 4. (a) A diagram of the substrate used in the pat cleavage assay. A 687 bp Pl DNA fragment (EcoRI-20) containing the pat site (site shown by open boxes) was end-labeled with [Y-~~P]ATP and polynucleotide kinase. Cleavage of this fragment at put generates 2 smaller fragments, 545 and 142 bp in size. (b) The fragments generated by pat cleavage. The preparation of cell extracts and the details of the pat cleavage assay are described in Materials and Methods. Samples were electrophoresed on a 5% acrylamide gel and the bands were visualized by autoradiography. Lanes: 1, no extract added; 2, NS3208; 3, pPAC3, 4, pPAC2; 5, pPAC4; 6, pPAC2 plus pPAC4; 7. pPAC16; 8, pPACl6 plus pPAC4.

when these two extracts are mixed they complement each other for cleavage activity (Fig. 4(b), lane 6). These results indicate that cleavage at the Pl pat site requires both the PacA and the PacB proteins. As mentioned above, cell extracts producing the 52,000 Mr PacA protein may also produce a 42,000-M, protein lacking the amino-terminal end of PacA. To ascertain whether the in vitro cleavage activity observed here with pPAC2 is attributed to the 52,000-M, PacA protein and not to any other protein present in the extract, an extract lacking the 52,000-M, protein (pPAC16) was tested either alone, or in combination with PacB, for its ability to cleave the pat site in vitro. Since the extract lacking the 52,000-M, PacA protein does not cleave pat,

either alone (Fig. 4(b), lane 7) or in the presence of PacB (Fig. 4(b), lane 8), our results indicate that the 52,000-M, PacA protein is required in conjunction with PacB to cleave the pat site. 4. Discussion (a) pacA and pacB genes The nucleotide sequence of the region of Pl DNA from the end of gene 10 to the end of the cl gene contains two open reading frames, pacA and pacB, that encode proteins necessary for cleavage of the Pl pat site. The upstream open reading frame, pacA, corresponds to a gene previously designated gene 9 (Scott, 1968) A number of phage mutants producing empty, unattached heads but normal

986

K. Skorupski et al.

tails contain amber mutations that have been assigned to this gene (Walker & Walker, 1983). Some of these mutants were later found to be de% cient in their ability to cleave PI DNA at pat (Sternberg & Coulby, 1987a). The inability of three of these amber mutations, amber 9.16, 43 and 131, to complement one another led to the suggestion that all three mapped to the one gene (Walker & Walker, 1983; Sternberg & Coulby, 1987a). Nucleotide sequencing of the DNA from all three PI mutants now clearly demonstrates that only amber 9.16 maps in gene 9 (pacA); the other two mutations, amber 43 and amber 131, map within the downstream gene pa&. One explanation for the failure of phage containing the amber 9.16 mutation to complement those containing the amber 43 or amber 131 mutations is that amber 9.16 is polar on the downstream pacB gene. (b) Expression of pacA and pacB The pacA and pacB genes are expressed relatively early in the phage growth cycle since pat cleavage is detectable within 10 to 15 minutes after PI infection (Sternberg & Coulby, 1987a). In addition pat cleavage is unaffected by the absence of the gene 10 product, which is involved in the control of late gene expression in PI (Walker & Walker, 1980, 1983). Since no obvious promoter sequences were identified within the nucleotide sequence presented here, it is likely that the pacA and pacB genes are expressed from a repressor-regulated promoter that lies immediately upstream of gene 10 (Lehnherr et al., 1991). Thus, pacA, pacB and gene 10 appear to be co-ordinately expressed during PI infection. The early expression of the pacase genes during infection suggests that these proteins may play a role in the viral life cycle in addition to their role in packaging. The T7 expression system used here to express pacA and pacB (Studier & Moffatt, 1986; Rosenberg et al., 1987), is an extremely powerful system for the expression of cloned genes. Its effectiveness is due in large part to the efficiency and selectivity of T7 RNA polymerase in transcribing genes with T7 promoters. The yields of the PacA and PacB proteins obtained with this system were surprisingly low, since the two proteins could be detected only by labeling with [35S]methionine in the presence of rifampicin. This low level of pacA and pacB expression suggests that these two genes are expressed relatively poorly during PI infection. Such low level expression may be a general characteristic of phage terminase genes. Low level expression of the terminase genes of lambda, T4 and T3 has been observed during infection with these phages (Murialdo et al., 1987; Rao & Black, 1988; Hamada et al., 1986). Replacement of the ribosomebinding sites of the lambda terminase genes with sites that more nearly match the consensus sequence has improved the expression of these genes (Murialdo et al., 1987). Similarly, we have discovered that replacement of the ribosome-binding sites of both pacA and pacB with the ribosome-

binding site of T7 gene 10 leads to a significant improvement in the expression of these two genes (our unpublished results). The pacA gene is expressed somewhat more etfcientl than pacB. Although the intensities of the two [7‘S]-labeled PacA and PacB proteins appear similar (Fig. 3(a) and (b), lane 3), the PacA protein contains seven methionine residues whereas the PacB protein contains 16 methionine residues. A similar pattern of expression has been observed with the lambda terminase proteins gpNu1 and gpA, and the T7 terminase proteins gp18 and gpl9. The first genes in each of these operons, Nul and 18, are expressed more efficiently than the second (Chow et al., 1987; White & Richardson, 1988).

(c) Function of PacA am? PacB Both the PacA and PacB proteins are necessary for cleavage of the PI pat site in vitro. Extracts from cells that produce both of these proteins cleave pat efficiently and extracts prepared from cells that express either protein alone complement each other for cleavage. By analogy with the many other ds DNA phages that have heteroligomeric terminase enzymes, it is likely that PacA and PacB are the two major subunits of the PI pacase enzyme. In lambda, P22, T7, T3 and T4, the large terminase subunit ranges from M, 60,000 to 75,000 in size. The PacB protein, with a sequenced molecular weight of 55.6 kDa, is similar in size. The small terminase subunits of these phages range from &Z, 10,000 to 30,000 in size. Since the sequenced molecular weight of the PacA protein is 45.2 kDa, the slightly larger size found for the PacA protein may indicate that this protein has additional enzymatic activities and/or roles in the phage life cycle in addition to pat cleavage. The positions of pacA and paeB on the PI genetic map are also analogous to the positions of the large and small subunits of the lambda, P22, T7 and T4 terminase enzymes. In all of these systems, the gene encoding the larger of the two subunits of the terminase enzyme lies immediately downstream from the gene encoding the small subunit. Although the specific roles of the PacA and PacB proteins in the pat-cleavage event are not yet known, it is tempting to speculate about their functions by drawing analogies with previously characterized phage terminase proteins. The small subunits of the lambda and P22 terminases, gpNu1 and gp3 respectively, apparently are involved in binding to the specific packaging recognition sequences on the DNA (Frackman et al., 1985; Jackson et al., 1982). Similarly, the PacA protein may have a role in DNA binding. The location of the pat-cleavage site within the gene encoding the smaller of the two P22 terminase proteins (Backhaus, 1985), is analogous to the situation observed in PI, and may reflect a similarity in function between gp3 and PacA. The large subunits of the lambda terminase, gpA, and the T3 terminase,

Pl

Genes Involved

gp19, appear to be involved in prohead binding (Frackman et al. 1984; Nakasu et al., 1983) and interactions of these proteins with ATP have been demonstrated (Higgins et al., 1988; Hamada et al., 1987). A-type or B-type ATP-binding consensus sequences have been implicated in the formation of adenine nucleotide binding domains within proteins (Walker et al., 1982), and A-type sequences have been identified in the large subunits of the lambda, T7 and T4 terminases (Guo et al., 1987). We examined the PacA and PacB amino acid sequences for evidence of either A-type or B-type ATP-binding an A-type consensus sequences, and identified sequence at the amino-terminal end of the PacB protein. The A-type sequence, A/G-X4-G-K-S/T, begins at amino acid residue 26 of the PacB protein. This sequence was not found in the PacA protein consensus sequence, and B-type the R/K-X3-G-X3-L, was not found in either the PacA or PacB amino acid sequences. The identification of an A-type ATP-binding consensus sequence within PacB suggests that this protein may interact with ATP. Despite the fact that many of the various phage terminase proteins have analogous roles in the DNA packaging process, these proteins do not show significant amino acid homology. A comparison of the amino acid sequences of PacA and PaeB with other phage terminase proteins did not reveal any these proteins. among obvious homology Furthermore, no significant nucleotide or amino acid sequence homology to PacA or PacB was found in the GenBank database. The important role of Dam methylation in the Pl packaging reaction (Sternberg & Coulby, 1990) raised the possibility that PacA or PacB might share structural features with the adenine methylase family of proteins (Chandrasegaran & Smith, 1988). However, a careful search failed to reveal significant homology between these sets of proteins. (d) Other PI proteins with possible roles in pat cleavage In addition to PacA and PacB, two other proteins have been identified on SDS-polyacrylamide gels from constructs carrying the pacA and pacB genes. The first of these proteins, a 20,000-Jf, protein designated PacC, is encoded within the C-terminal end of the pacB gene and translated in the same reading frame as PacB. The relationship between pacB and pa& appears to be similar to that of the C and Nu3 genes of lambda since Nu3 is encoded within the C-terminal end of the C gene (Shaw & Murialdo, 1980). At present, the role of the PacC protein in pat cleavage, if any, is not known. One possibility is that PacC has an ancillary role in the packaging reaction analogous the 14 kDa Fl protein of lambda. The Fl protein is a non-capsid protein which appears to be involved in facilitating the interaction of proheads with the terminaseDNA complex (Davidson & Gold, 1987; Becker et al., 1988). A second protein, which is 42,000-M, in

987

in Pl pat Cleavage

size, also has been identified on SDS-polyacrylamide gels and may be produced from an internal start codon that lies within the pacA gene, Although this smaller protein lacks POX cleavage activity, the possibility that it might have a physiological role in the cleavage process has not been eliminated. The purification and further characterization of the Pl pacase proteins should provide new insights into the mechanism of DNA packaging and add to our understanding of this complex, yet fundamental system. Further biochemical characterization of these proteins should also lead to an improved efliciency of DNA packaging in the Pl DNA cloning system. We thank Hansjijrg Lehnherr for communicating results prior to publication, Ken Abremski for assistance with the computer analyses and for critical reading of the manuscript, and Marvin Kendall for synthesis of the oligonucleotides. The work reported in this manuscript was supported by National Institutes of Health grant ROl-GM42952-02.

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Chow, S., Daub, E. & Murialdo, H. (1987). The overproduction of DNA terminase of coliphage lambda. Gene, 60, 277-289. Davidson, A. & Gold, M. (1987). A novel in vitro DNA packaging system demonstrating a direct role for the bacteriophage 2 FI gene product. Virology, 161, 305-314. Davis, R. W., Botstein, D. & Roth, J. R. (1980). Advanced Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Dennert, G. & Henning, V. (1968). Tyrosine-incorporating amber suppressors in Escherichia coli K12. J. Mol. Biol. 33, 327-329. Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive set of sequence analysis programs for the V,4X. Nucl. Acids Res. 12, 387-395. Frackman, S., Siegele, D. A. & Feiss, M. (1984). A functional domain of bacteriophage 2 terminase for prohead binding. J. Mol. Biol. 180, 283-300. Frackman, S., Siegele, D. A. & Feiss, M. (1985). The terminase of bacteriophage 1. Functional domains for cosB binding and multimer assembly. J. Mol. Biol. 183, 225-238. Gough, J. A. & Murray, N. E. (1983). Sequence diversity among related genes for recognition of specific targets in DNA molecules. J. Mol. Biol. 166, I-20. Guo, P., Peterson, C. & Anderson, D. (1987). Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage 429. J. Mol. Biol. 197, 229-236. Hamada, K., Fujisawa, H. & Minagawa, T. (1986). Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging. Virology, 151, 11&118. Hamada, K., Fujisawa, H. & Minagawa, T. (1987). Characterization of ATPase activity of a defined in vitro system for packaging of bacteriophage T3 DNA. Virology, 159, 244-249. Hashimoto, C. & Fujisawa, H. (1988). Packaging and transduction of non-T3 DNA by bacteriophage T3. ViroZogy, 166, 432-439. Higgins, R. R., Lucko, H. J. & Becker, A. (1988). Mechanism of cos DNA cleavage by bacteriophage 1 terminase: multiple roles of ATP. Cell, 54, 765-775. Hohn, B. (1983). DNA sequences necessary for packaging of bacteriophage 1 DNA. Proc. Nat. Acad. Sci., U.S.A. 80, 74567460. Huynh. T. V., Young, R. A. & Davis, R. W. (1985). Constructing and screening cDNA libraries in 1 gtI0 and 1 gtll. In DNA Cloning (Glover, D. M., ed.), vol. I, pp. 49-78, IRL Press Ltd, Oxford, England. Jackson, E. N., Laski; F. & Andres, C. (1982). Bacteriophage P22 mutants that alter the specificity of DNA packaging. J. Mol. Biol. 154, 551-563. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685. Lehnherr, H., Guidolin, A. & Arber, W. (1991). The bacteriophage PI gene 10 encodes a trans activating factor required for late gene expression. J. Bacterial. 173, 6438-6445. Messing, J. (1983). New Ml3 vect,ors for cloning. Methods Enzymol. 101, 20-78. Murialdo, H.. Davidson, A., Chow, S. & Gold, M. (1987). The control of 1 DNA terminase synthesis. Nucl. Acids Res. 15, 119140. Nakasu, S., Fujisawa, H. & Minagawa, T. (1983). Role of geue 8 product in morphogenesis of bacteriophage T3. Virology, 127, 124-133.

et al. Osborne, F. A., Stovall, S. R. & Baumstark, B. R. (1989). The cl genes of PI and P7. Nucl. Acids Res. 17, 7671-7680. Rao, V. B. & Black, L. W. (1985). Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product. CeZl, 42, 967-977. Rao, V. B. & Black, L. W. (1988). Cloning, overexpression and purification of the terminase proteins gpl6 and gp17 of bacteriophage T4: construction of a defined in vitro DNA packaging system using purified terminase proteins. J. Mol. Biol. 200, 475-488. Rosenberg, A. H., Lade, B. N.. Chui, D., Lin, S.-W.. Dunn, J. J. & Studier, F. W. (1987). Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene, 56, 125-135. Rosner. J. L. (1972). Formation, induction and curing of bacteriophage Pl lysogens. Virology, 48, 679-689. Sambrook, ,J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning, 2nd edit. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger. F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Pruc. Nat. Acad Sci., U.S.A. 74, 546335467. Sayers, J. R., Schmidt, W. & Eckstein, F. (1988). 5’-3’ exonucleases in phosphorothioat,e-based oligonucleotide-directed mutagenesis. Nucl. Acids Res. 16. 791-802. Scott, J. R. (1968). Genetic studies on bacteriophage Pl. Virology, 36, 564-574. Shaw. J. E. & Murialdo, H. (1980). Morphogenetic genes (I and Nu3 overlap in bacteriophage 1. Nature (London), 283, 30-35. Silhavy. T. J., Berman, M. L. $ Enquist, L. W. (1984). Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sternberg, N. (1990). Bacteriophage PI cloning system for the isolation, amplification. and recovery of DNA fragments as large as 100 kilobase pairs. Proc. Nat. Acad. Sci., U.S.A. 87, 103-107. Sternberg, N. & Coulby, J. (1987a). Recognition and cleavage of the bacteriophage PI packaging site (pat). I. Differential processing of the cleaved ends in vivo. J. Mol. Biol. 194, 453468. Sternberg, N. & Coulby, J. (1987b). Recognition and cleavage of the bacteriophage PI packaging site (pat). II. Functional limits of pat and location of pat cleavage termini. J. Mol. Biol. 194, 469-479. Sternberg, N. & Coulby, J. (1990). Cleavage of the bacteriophage PI packaging site (pat) is regulated by adenine methylation. Proc. Nat. Acad. Sci., ~T.8.A. 87, 80708074. Sternberg, N., Tiemeier, D. & Enquist, 1~. (1977). In r&o packaging of a L Dam vector containing EcoRI DNA fragments of Escherichia coli and phage Pl Gene, 1. 255-280. Sternberg, N., Ruether, J. & deRie1, K. (1990). Generation of a 50,000-member human DNA library with an average DNA insert size of 75-100 kbp in a bacteriophage PI cloning vector. The New Biologist, 2. I51-162. Streisinger, C., Emrich, J. & Stahl, M. M. (1967). Chromosome structure in phage T4. III. Terminal redundancy and length determination. Proc. Nat. Acud. Sci., U.S.A. 57, 292-295. Stretton, A. 0. W., Kaplan, S. & Brenner, S. (1966). Nonsense codons. Cold Spring Harbor Symp. Quant. Biol. 31. 173-179. Studier, F. W. & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct, selective high-

Pl

Genes Involved

level expression of cloned genes. J. Mol. Biol. 189, 113-130. Tye, B.-K., Huberman, J. A. & Botstein, D. (1974). Non-random circular permutation of phage P22 DNA. J. Mol. Biol. 85, 501-527. Walker, D. H., Jr 8: Walker, ,J. T. (1975). Genetic studies of coliphage Pl. I. Mapping by use of prophage deletions. J. Viral. 16, 525-534. Walker, J. T. & Walker, D. H., Jr (1980). Mutations in coliphage Pl affecting host cell lysis. J. Viral. 35, 61!&530. Edited

in Pl

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Walker, J. T. & Walker, D. H., Jr (1983). Coliphage Pl morphogenesis: analysis of mutants by electron microscopy. J. Viral. 45, 1118-1139. Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982). Distantly related sequences in the alpha subunits and beta subunits of ATP myosin kinases and other ATP requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945-952. White, J. H. & Richardson, C. C. (1988). Gene 19 of bacteriophage T7. J. Biol. Chem. 263. 2469-2476.

by M. E. Gottesman